CN1192447A - Adsorption resin method for conectrating and separating vegetable baudy glucoside from stevioside - Google Patents

Adsorption resin method for conectrating and separating vegetable baudy glucoside from stevioside Download PDF

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CN1192447A
CN1192447A CN 98104776 CN98104776A CN1192447A CN 1192447 A CN1192447 A CN 1192447A CN 98104776 CN98104776 CN 98104776 CN 98104776 A CN98104776 A CN 98104776A CN 1192447 A CN1192447 A CN 1192447A
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resin
effluent
stevioside
glycosides
hours
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CN 98104776
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CN1078217C (en )
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何炳林
陈天红
张杨
史作清
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南开大学
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Abstract

The present invention designs and synthesizes a series of high selective macroporous polymeric adsorbent. By using the selective adsorption of macroporous polymeric adsorbent to separate stevioside containing high glycoside A from the leaf of stevia rebaudian and through recrystallization stevioside glycoside A producet with purity higher than 90% is obtd.

Description

吸附树脂法从甜菊糖中富集、分离菜鲍迪甙A Adsorption resin enriched from stevia, isolated glycosides A food Bowditch

本发明属大孔吸附树脂的合成及其应用。 The present invention belongs to the synthesis and application of macroporous resin.

甜菊糖是从甜叶子中提取出来的一种高甜度、低热值、非糖、非营养型安全可靠的天然甜味剂,它主要由8种已知糖甙组成。 Stevia is extracted from the leaves of the sweet kind of high sweetness, low calorific value, non-sugar, non-nutritive sweeteners safe and reliable natural, it mainly consists of eight known species of glycoside composition. 其名称和化学结构如表1所示[1]。 As its name and the chemical structure shown in Table 1 [1]. (1.Hansib JR,DeOliveira BH,Stevioside and related sweet diterpenoid glycosides,Natural Product Reports,1993,301-309)表1甜叶菊总糖甙中各组份结构及相对于叶含量 (1.Hansib JR, DeOliveira BH, Stevioside and related sweet diterpenoid glycosides, Natural Product Reports, 1993,301-309) Table 1 Total Stevia glycosides of each component in the structure and relative Leaves

G葡萄糖基Rh-鼠李糖基其中甜菊甙(Stevioside)、莱鲍迪甙A(RebaudiosideA)和莱鲍迪甙C(Rebaudioside c)在总糖中的相对含量较高,也是影响甜菊糖味质的主要成份。 G glucosyl-rhamnosyl wherein Rh- stevioside (Stevioside), rebaudioside A (RebaudiosideA) and rebaudioside C (Rebaudioside c) relatively high content of total sugars, but also affect the taste quality Stevia the main ingredients. S甙的甜度是蔗糖的270-280倍,但它呈味速度慢,带有一定的不良余味。 S glycosides sweetness of 270-280 times that of sugar, but it is slow taste, with a certain bad aftertaste. (甙的甜度不到蔗糖的50倍,且有较强的后苦味及不良余味。C甙的甜度不到蔗糖的50倍,且有较强的后苦味及不良余味。A甙的甜度最高,大于蔗糖的300倍,而且味质也最好,不含任何不良余味,是一种最为理想的天然甜味剂。提取分离高A甙含量的甜菊糖产品是近年来国际、国内甜菊糖生产工业的研究热点。然而,由于这几种糖甙具有相同的甙元,其结构与分子极性都非常相近,因此很难用常规手段将其分离,特别是分离高纯度、高品质的A甙产品。迄今为止,有关甜菊糖各组份分离报导的主要手段有:高效液相色谱法(HPLC)[2-5][2.Dobberstein RH,Almed MS,Extraction,separationand recovery of diterpene glycosides fromStevia rebaudiana plants.US 4,361,697,1982;3.AhmedM.S.,Dobberstein RH,Stevia rebaudiana.II High-performance“quid chromatographicseparation and quantitation of stevioside,rebaudioside A and rebaud (Glycosides sweetness less than 50 times that of sucrose, and the strong bitter aftertaste and adverse .C glycosides sweetness less than 50 times that of sucrose, and the strong bitter aftertaste and poor sweet glycosides .A the highest degree, 300 times greater than sucrose, but also taste the best quality, does not contain any bad aftertaste, it is one of the most ideal natural sweetener. a high glucosinolate content extraction separation of stevia products in recent years international and domestic stevia sugar production industry research focus. However, since these types of glycosides have the same aglycone, its structure and polar molecules are very similar, it is difficult to be separated by conventional means, particularly the separation of high-purity, high-quality . a product glycoside to date, the primary means of separation of stevioside reported about the components are: high performance liquid chromatography (HPLC) [2-5] [2.Dobberstein RH, Almed MS, Extraction, separationand recovery of diterpene glycosides fromStevia rebaudiana plants.US 4,361,697,1982;. 3.AhmedM.S, Dobberstein RH, Stevia rebaudiana.II High-performance "quid chromatographicseparation and quantitation of stevioside, rebaudioside A and rebaud ioside CJChromatogr,1982,236(2):523-526;4.Ahmed MS,Dobberstein RH,Stevia rebaudiana.II High-performanceliquid chromatographic separation and quantitation of rebaudioside B,Dand E,dulcoside A andsteviolbioside.J.Chromatogr.,1982,245(3):373~376;5.Makapugay HC,Nanayakkara NPD,Kinghorn AD,Improved high-performance liquid chromatographic separation of the steviarebaudiana sweet diterpene glycosides using linear gradient elution,J.Chromatogr.,1984,283,390~395]、薄层色谱法(TLC)[6-7][6.Metivier J,Viana AM,Determination of microgramquantities of stevioside from leaves of Stevia rebaudiana Bert.bytwo-dimensional thin layerchromatography.J.Exp.Bot.,1979,30(117):805~810;7 Sherma J.,Norfolk E.,Quantitative TLCdetermination of stevioside and rebaudioside A in beverages J Liq.Chromatogr.,1992,15(17):2981~2988]、滴液逆流色谱法[8][8.Kinghorn AD,Nanayakkara NPD,Soejarto DDetal.,Potential sweetening agents of plant or ioside CJChromatogr, 1982,236 (2): 523-526; 4.Ahmed MS, Dobberstein RH, Stevia rebaudiana.II High-performanceliquid chromatographic separation and quantitation of rebaudioside B, Dand E, dulcoside A andsteviolbioside.J.Chromatogr, 1982. , 245 (3):. 373 ~ 376; 5.Makapugay HC, Nanayakkara NPD, Kinghorn AD, Improved high-performance liquid chromatographic separation of the steviarebaudiana sweet diterpene glycosides using linear gradient elution, J.Chromatogr, 1984,283,390 ~ 395], thin layer chromatography (TLC) [6-7] [6.Metivier J, Viana AM, Determination of microgramquantities of stevioside from leaves of Stevia rebaudiana Bert.bytwo-dimensional thin layerchromatography.J.Exp.Bot., 1979 , 30 (117): 805 ~ 810; 7 Sherma J., Norfolk E., Quantitative TLCdetermination of stevioside and rebaudioside A in beverages J Liq.Chromatogr, 1992,15 (17): 2981 ~ 2988], dropping countercurrent chromatography. method [8] [8.Kinghorn AD, Nanayakkara NPD, Soejarto DDetal., Potential sweetening agents of plant or gin I Purification of Stevia rebaudiana sweetconstituents by droplet countercurrent chromatography.J.Chromatogr.,1982,237(3):478-483]、毛细管电泳法[9-10][9.Liu J.,Li SFY,Separation and determination of Stevia sweeteners bycapillary electrophoresis and high performance liquid chromatogr.,J.Liq.Chromatogr.,1995,18(9):1703-1719;10.Mauri P.,Catalano G.,Gardana C.,Pietta P.,Analysis of Stevia glycosides bycapillary electrophoresis.Electrophoresis,1996,17(2):367~371]、超临界萃取法[11-12][11.KienleUdo,Process for the preparation of a natural sweetening agent from Stevia rebaudiana and its use.EP 335,265,1989;12.Tan S.,Shibuta Y.,Tanaka O.,Isolation of sweetener from SteviarebaudianaJP63,177,764,1988]等,但这些方法的可处理量小,都不适合开发工业化生产。 gin I Purification of Stevia rebaudiana sweetconstituents by droplet countercurrent chromatography.J.Chromatogr, 1982,237 (3):. 478-483], capillary electrophoresis [9-10] [9.Liu J., Li SFY, Separation and determination of Stevia sweeteners bycapillary electrophoresis and high performance liquid chromatogr, J.Liq.Chromatogr, 1995,18 (9):.. 1703-1719; 10.Mauri P., Catalano G., Gardana C., Pietta P., Analysis of Stevia glycosides bycapillary electrophoresis.Electrophoresis, 1996,17 (2): 367 ~ 371], supercritical extraction [11-12] [11.KienleUdo, process for the preparation of a natural sweetening agent from Stevia rebaudiana and its use.EP 335,265,1989; 12.Tan S., Shibuta Y., Tanaka O., Isolation of sweetener from SteviarebaudianaJP63,177,764,1988] and the like, but small amounts of these methods may be processed, it is not suitable for the development of industrial production. 近两年来,日本出现了从一种特殊的高A甙含量的甜菊叶中通过重结晶法提取分离A甙的报导[13-14][13.Morita T.,Nishimura M,Ishikawa H.,Manufacture of a sweetener difficult to dissolve inwater from Stevia extract.JP 07,177,802,1995;14.Katanami T.,Kitatsume M.,Prep.of Steviasweetener.JP 07,143,860,1995],其产品已开始打入国际市场。 The last two years, there have been reported in Japan, a special extract from Stevia glycosides A high content of A glycosides isolated by recrystallization method [13-14] [13.Morita T., Nishimura M, Ishikawa H., Manufacture of a sweetener difficult to dissolve inwater from Stevia extract.JP 07,177,802,1995; 14.Katanami T., Kitatsume M., Prep.of Steviasweetener.JP 07,143,860,1995], its products have begun to enter the international market. 但他们所选用的甜菊叶原料较难获得,A甙含量大于80%的甜菊糖产品成本高于普通甜菊糖的4-5倍。 But they are the choice of raw materials difficult to obtain stevia, Stevia glycosides A content costs more than 80% higher than 4-5 times that of ordinary stevioside.

利用树脂吸附的弱选择性特点分离甜菊糖的研究目前尚未见国内外有关文献和专利报导。 Utilization Adsorption characteristics of weak selective separation of stevioside and has yet to see the patent literature reports at home and abroad. 本发明的目的是设计合成了系列具有高选择性大孔吸附树脂,首次尝试了利用大孔吸附树脂选择性吸附的作用通过简单可行,易于工业化的手段从高S甙甜菊糖中分离出富集A甙的甜菊糖产品,再经进一步纯化,得到纯度大于90%的A甙产品。 Object of the present invention is to devise a series of highly selective synthesis of macroporous resin, the effect of the first attempt to use macroporous resin selectively adsorbed by a simple and feasible means of easily separated from the high industrialized glycosides Stevioside enriched S a stevia glycosides, and then further purification, to give a glycoside product purity greater than 90%.

本发明的内容主要有树脂的设计合成;树脂法分离富集A甙工艺,以及A甙的进一步纯化。 The present invention is designed mainly of synthetic resin; A purified isolated further enriched glycosides glycosides A process, method and a resin. 树脂的合成方法如下:于三口瓶中将工业二乙烯苯、4--乙烯基吡啶和过氧化苯甲酰混合均匀,加入混合致孔剂烷基苯与液体石蜡。 The method of synthetic resin as follows: to a three-necked flask were mixed uniformly industrial divinylbenzene, 4-- vinyl pyridine, and benzoyl peroxide, mixed porogen were added liquid paraffin and alkylbenzene. 加入NaCl水溶液,使水油相体积比控制在一定范围内,加入聚乙烯醇,做表面活性剂,开动搅拌,控制搅拌速度,使小球的粒径分布在80-200目之间,升温至80℃,恒温反应2小时,继续升温至90℃,恒温反应4小时。 NaCl solution was added, the aqueous phase volume ratio of the oil is controlled within a certain range, addition of polyvinyl alcohol, surface active agents, Stirring, agitation speed control, so that the particle size distribution of the beads between 80-200 mesh, warmed to 80 ℃, the reaction temperature for 2 hours and continue to heat to 90 ℃, reacting at the temperature for 4 hours. 最后升温至95-100℃煮球2小时,使聚合完全。 Finally, after heating to 95-100 deg.] C ball and cook for 2 hours to complete the polymerization. 所得树脂用热水充分洗涤,凉干后分别用乙醇、石油醚各抽提8小时,室温下减压干燥备用。 The resulting resin was washed well with water, dry it with ethanol, respectively, each of the petroleum ether extraction for 8 hours and dried under reduced pressure at room temperature for standby.

树脂法分离富集A甙包括树脂的活化及A甙的富集,树脂的活化方法如下:称取一定量的树脂用适量乙醇溶胀过夜,然后逐渐过渡到水相。 Enrichment A resin was isolated and activated include glycosides glycosides A resin enrichment, the resin activation method as follows: weighed amount of resin with an appropriate amount of ethanol to swell overnight, then gradually transition to the aqueous phase. 准确量取一定体积的湿树脂并将其转入内径为0.8~1.0cm树脂柱中,以1BV/h的流速使20-30ml 5%的稀HCl溶液缓慢流过树脂床、水洗至中性备用。 The exact amount of a certain volume of the wet resin was transferred to an inside diameter of 0.8 ~ 1.0cm resin column, at 1BV / h flow rate of 20-30ml 5% so that a solution of dilute HCl was slowly flows through the resin bed, washed with water until neutral standby .

A甙的富集可用下列两个方法:其中一个方法是配制一定浓度的甜菊糖水溶液,使其以一定流速缓慢流过树脂床。 A glucoside enriched available the following two methods: One method is to formulate an aqueous solution of a certain concentration of stevioside, it slowly flows at a constant flow rate of the resin bed. 此时树脂对甜菊糖分子产生吸附与选择性吸附作用。 At this time, resin adsorption and generating selective adsorption of molecules of stevioside. 检测流出液,自泄漏点起每10ml或20ml为一组份等体积接收流出液,并用HPLC进行监测,直至流出液中A甙含量小于S甙。 Detecting the effluent from leaks or from 20ml to 10ml per group received equal volumes effluent, and monitored by HPLC, and until the effluent A glucosinolate content of less than S glycosides. 合并流出液浓缩至干燥,所得甜菊糖中A甙含量高于S甙。 The combined effluent was concentrated to dryness, the resulting steviol glycoside content is greater than A S glycosides. 另一个富集A甙的方法是将高S甙甜菊糖用甲醇重结晶,结晶母液浓缩至干燥。 A further method of enriching a high S glycosides are glycosides stevioside was recrystallized from methanol, crystallization mother liquor was concentrated to dryness. 然后将母液糖配成一定浓度的水溶液,使其以一定流速缓慢流过树脂床。 The mother liquor was then formulated into an aqueous solution of a certain concentration of sugar, so a constant flow slowly through the resin bed. 检测流出液,自泄漏点起每10ml或20ml为一组份等体积接收流出液,并用HPLC进行监测,直至流出液中的杂质含量与原母液接近为止。 Detecting the effluent from leaks or from 20ml to 10ml per group received equal volumes effluent, and monitored by HPLC, and the effluent until the impurity content of the mother liquor of the original close up. 合并流出液,浓缩至干燥,所得甜菊糖中A甙含量高于S甙,并且杂质含量低于原母液。 The combined effluent was concentrated to dryness, the resulting steviol glycoside content is greater than A S glycosides, and the impurity content is less than the original mother liquor.

A甙的进一步纯化方法:将用树脂法处理得到的高A甙含量的甜菊糖产品,用含水量为0~50%的甲醇进行重结晶。 A method for further purification of saponins: the processing method obtained with a high resin content of Stevia glycosides A, with a water content of 0-50% is recrystallized from methanol.

通过上述方法得到的高A甙含量甜菊糖产品,用含水量为0~50%的甲醇进行重结晶,通过控制重结晶条件可以得到A甙含量大于90%的结晶产品熔点为227~229℃。 Obtained by the above method A high content of Stevia glycosides, 0-50% methanol recrystallized moisture content can be obtained A saponin content greater than 90% of the crystalline product melting at 227 ~ 229 ℃ by controlling the recrystallization condition.

实例1于三口瓶中将工业二乙烯苯95克、4-乙烯基吡啶5克和过氧化苯甲酰1克,混合均匀后加入混合致孔剂烷基苯与液体石蜡,总重量为200克,其中烷基苯与石蜡的重量比为2∶1。 Example 1 of the three-neck flask 95 g of industrial divinylbenzene, 5 g of 4-vinyl pyridine and 1 g of benzoyl peroxide, mixed uniformly mixed porogen were added liquid paraffin and alkylbenzene, a total weight of 200 grams , alkylphenyl wherein the weight ratio of 2 to paraffin wax. 加入NaCl水溶液,使水油相体积比控制在5∶1,并加入0.5%的聚乙烯醇作表面活性剂。 NaCl solution was added, the aqueous phase volume ratio of oil in 5:1 control, and 0.5% polyvinyl alcohol as a surfactant. 开动搅拌,控制搅拌速度,使小球粒径分布在80~200目之间。 Stirring, agitation speed control, so that the ball size distribution between 80 to 200 mesh. 以5℃/10min的速度匀速缓慢升温至80℃,恒温反应2小时。 At a rate of 5 ℃ / 10min constant speed slowly warmed to 80 ℃, the reaction temperature for 2 hours. 继续升温至90℃,恒温反应4小时。 Continue to heat up to 90 ℃, reacting at the temperature for 4 hours. 最后升温至95~100℃煮球2小时,使聚合完全。 Finally, after heating to 95 ~ 100 ℃ cook balls for 2 hours to complete the polymerization. 所得树脂用热水充分洗涤,凉干后分别用乙醇、石油醚各抽提8小时,室温下减压干燥备用。 The resulting resin was washed well with water, dry it with ethanol, respectively, each of the petroleum ether extraction for 8 hours and dried under reduced pressure at room temperature for standby.

称取一定量的树脂用适量乙醇溶胀过夜,然后逐渐过渡到水相。 Weighed amount of resin with an appropriate amount of ethanol to swell overnight, then gradually transition to the aqueous phase. 准确量取20ml湿树脂并将其转入内径为0.8~1.0cm树脂柱中,以1BV/h的流速使20~30ml 5%的稀HCl溶液缓慢流过树脂床,然后水洗至中性。 20ml of wet resin was weighed accurately and transferred into an inner diameter of 0.8 ~ 1.0cm resin column, at 1BV / h so that a flow rate of 20 ~ 30ml 5% HCl solution was slowly diluted to flow through the resin bed, and then washed with water until neutral. 再以1BV/h的流速使20~30ml 1N的NaOH溶液缓慢流过树脂床,水洗至中性备用。 Then at 1BV / h at a flow rate that the solution 20 ~ 30ml 1N NaOH slowly flows through the resin bed, washed with water until neutral standby.

配制4mg/ml的甜菊糖水溶液,使其以1BV/h的流速缓慢流过树脂床。 Formulated 4mg / ml aqueous solution of stevioside, so to 1BV / h of flow rate through the resin bed slowly flowing. 此时树脂对甜菊糖分子产生选择性吸附作用。 In this case the resin produced selective adsorption of molecules of stevioside. 检测流出液,流出液达380ml开始泄漏,自泄漏点起每20ml为一组分,等体积接收流出液,并用HPLC进行监测,直至500ml后流出液中A甙含量小于S甙。 Detector effluent, the effluent of 380ml begins to leak, the leak from the starting point to a 20ml each component receiving volume of effluent and the like, and monitored by HPLC, and after 500ml until the effluent A glucosinolate content of less than S glycosides. 合并流出液,浓缩至干燥,得到高A甙含量甜菊糖约200mg,其中A甙与S甙的含量比约为(2.8~1.0)∶1。 The combined effluent was concentrated to dryness to obtain a high content of steviol glycosides A about 200mg, and S wherein the content of glycosides glycosides A ratio of about (2.8 to 1.0) to 1. 树脂可用70%乙醇再生,用水洗净后可反复使用。 Resin can be regenerated with 70% ethanol, washed with water can be used repeatedly. 图1给出原糖液与流出液的HPLC谱图。 Figure 1 shows raw sugar and HPLC profile of the effluent.

实例2于三口瓶中将工业二乙烯苯97克、4-乙烯基吡啶3克和1克过氧化苯甲酰混合均匀、加入混合致孔剂烷基苯与液体石蜡,总重量为200克,其中烷基苯与石蜡的重量比为5∶1。 Example 2 A three-necked flask to the 97 g of industrial divinyl benzene, 4-vinyl pyridine 3 g and 1 g of benzoyl peroxide were mixed uniformly, added to the mixed porogen alkylbenzene and liquid paraffin, a total weight of 200 grams, wherein the weight ratio of paraffin and alkylbenzene 5:1. 以NaCl水溶液做为分散剂,水油相体积比控制在5∶1,并加入0.5%的聚乙烯醇做为表面活性剂。 As dispersing agents in aqueous NaCl, the volume ratio of water to oil phase control 5:1, and 0.5% polyvinyl alcohol as a surfactant. 开动搅拌,控制搅拌速度,使小球粒径分布在80~200目之间。 Stirring, agitation speed control, so that the ball size distribution between 80 to 200 mesh. 以5℃/10min的速度匀速缓慢升温至80℃,恒温反应2小时。 At a rate of 5 ℃ / 10min constant speed slowly warmed to 80 ℃, the reaction temperature for 2 hours. 继续升温90℃,恒温反应4小时。 Continue to heat 90 ℃, reacting at the temperature for 4 hours. 最后升温至90~100℃煮球2小时,使聚合完全。 Finally, after heating to 90 ~ 100 ℃ cook balls for 2 hours to complete the polymerization. 所得树脂用热水充分洗涤,凉干后分别用乙醇、石油醚各抽提8小时,室温下减压干燥备用。 The resulting resin was washed well with water, dry it with ethanol, respectively, each of the petroleum ether extraction for 8 hours and dried under reduced pressure at room temperature for standby.

称取一定量的树脂用适用乙醇溶胀过夜,然后逐渐过渡到水相。 Weighed amount of resin with suitable alcohol to swell overnight, then gradually transition to the aqueous phase. 准确量取10ml湿树脂并将其转入内径为0.8~1.0cm树脂柱中,以1BV/h的流速使20~30ml 5%的稀HCl溶液缓慢流过树脂床,然后水洗至中性。 10ml of wet resin was weighed accurately and transferred into an inner diameter of 0.8 ~ 1.0cm resin column, at 1BV / h so that a flow rate of 20 ~ 30ml 5% HCl solution was slowly diluted to flow through the resin bed, and then washed with water until neutral. 再以1BV/h的流速使20~30ml 5%的稀HCl溶液缓慢流过树脂床,然后水洗至中性备用。 Then at 1BV / h so that a flow rate of 20 ~ 30ml 5% HCl solution was slowly diluted to flow through the resin bed, and then washed with water until neutral standby.

称取20克甜菊糖,用250ml甲醇溶解,于3~5℃冷却结晶48小时。 Weigh 20 g stevia, dissolved with 250ml of methanol, the cooling crystallization 3 ~ 5 ℃ 48 hours. 过滤,并将母液浓缩至干燥。 Filtered and the mother liquor was concentrated to dryness. 将母液糖配制成4mg/ml的甜菊糖水溶液,使其以1BV/h的流速缓慢流过树脂床。 The mother liquor was formulated as sugar Stevioside aqueous 4mg / ml, making it at 1BV / h of flow rate through the resin bed slowly flowing. 此时,树脂对甜菊糖及其杂质分子产生选择性吸附作用。 At this time, the resin is generated and the selective adsorption of impurities on molecular stevioside. 检测流出液,达185ml后开始泄漏,自泄漏点起每20ml为一组份等体积接收流出液,并用HPLC进行监测,445ml以后流出液中的杂质含量开始显著增高,并且A甙与S甙的比值也基本接近母液糖。 Detecting effluent, after 185ml of began to leak from the leak from each 20ml of a set of equal volumes receiving effluent, and monitored by HPLC, and after 445ml start impurity content of the effluent was significantly higher, and A glycoside and S-glycosides basically sugar liquor ratio close. 停止收集,合并流出液,浓缩至干燥,得到高A甙含量甜菊糖约610mg,其中A甙与S甙的含量比约为(8.1~1.7)∶1。 Stop collecting the combined effluent was concentrated to dryness to obtain a high content of steviol glycosides about 610mg A, wherein the A and saponin glycosides S ratio of about (8.1 to 1.7) to 1. 图2给出母液糖与流出液糖的HPLC谱图。 Figure 2 shows the HPLC profile of the mother liquor effluent liquid sugar and sugar.

实例3于三口瓶中将工业二乙烯苯50克、4-乙烯基吡啶10克和过氧化苯甲酰0.8克混合均匀,加入混合致孔剂烷基苯与液体石蜡,总重量为60克,其中烷基苯与石蜡的重量比为1∶1。 Example 3 To a three-neck flask to 50 g of industrial divinyl benzene, 4-vinyl pyridine and 10 g of benzoyl peroxide mixed 0.8 g, was added and mixed porogen alkylbenzene liquid paraffin, a total weight of 60 grams, alkylphenyl wherein the weight ratio of 1 to paraffin wax. 加入NaCl水溶液,使水油相体积比控制在4∶1,并加入0.8%的聚乙烯醇做为表面活性剂。 NaCl solution was added, the aqueous phase volume ratio of oil in 4:1 control, and 0.8% polyvinyl alcohol as a surfactant. 开动搅拌,控制搅拌速度,使小球粒径分布在80~200目之间。 Stirring, agitation speed control, so that the ball size distribution between 80 to 200 mesh. 以5℃/10min的速度匀速缓慢升温至80℃,恒温反应2小时。 At a rate of 5 ℃ / 10min constant speed slowly warmed to 80 ℃, the reaction temperature for 2 hours. 继续升温90℃,恒温反应4小时。 Continue to heat 90 ℃, reacting at the temperature for 4 hours. 最后升温至90~100℃煮球2小时,使聚合完全。 Finally, after heating to 90 ~ 100 ℃ cook balls for 2 hours to complete the polymerization. 所得树脂用热水充分洗涤,凉干后分别用乙醇、石油醚各抽提8小时,室温下减压干燥备用。 The resulting resin was washed well with water, dry it with ethanol, respectively, each of the petroleum ether extraction for 8 hours and dried under reduced pressure at room temperature for standby.

称取一定量的树脂,用适量乙醇溶胀过夜,然后逐渐过渡到水相。 Weighed amount of resin with an appropriate amount of ethanol to swell overnight, then gradually transition to the aqueous phase. 准确量取10ml湿树脂并将其转入内径为0.8~1.0cm树脂柱中,以1BV/h的流速使20~30ml 5%的稀HCl溶液缓慢流过树脂床,然后水洗至中性。 10ml of wet resin was weighed accurately and transferred into an inner diameter of 0.8 ~ 1.0cm resin column, at 1BV / h so that a flow rate of 20 ~ 30ml 5% HCl solution was slowly diluted to flow through the resin bed, and then washed with water until neutral. 再以1BV/h的流速使20~30ml 1N的稀HCl溶液缓慢流过树脂床,并水洗至中性备用。 Then at 1BV / h flow rate of lean of 20 ~ 30ml 1N HCl solution slowly flows through the resin bed, and washed with water until neutral standby.

配制4mg/ml的甜菊糖水溶液,使其以1BV/h的流速缓慢流过树脂床。 Formulated 4mg / ml aqueous solution of stevioside, so to 1BV / h of flow rate through the resin bed slowly flowing. 此时,树脂对甜菊糖分子产生选择性吸附作用。 In this case, the resin to produce selective adsorption of molecules of stevioside. 检测流出液,120ml开始泄漏,自泄漏点起每10ml等体积接收流出液,并用HPLC进行监测,直至245ml以后流出液中A甙与S甙基本相等。 Effluent is monitored, 120ml begin to leak, the leak from the starting point of each received an equal volume of 10ml effluent, and monitored by HPLC, and after 245ml until the effluent A S glycosides and glycoside is substantially equal. 合并流出液,浓缩至干燥,得到高A甙含量甜菊糖约135mg。 The combined effluent was concentrated to dryness to obtain a high content of steviol glycosides A to about 135mg.

实例4准确称取5克用树脂法处理得到的高A甙含量甜菊糖产品,用含水量为10%的甲醇溶液加热溶解,过滤,冷却至3~5℃进行重结晶,可以得到A甙含量约为91-93%的结晶产品约2~2.5克,熔点为219~221℃。 Example 4 A high accurately weighed glucosinolate content Stevia treated with 5 g of the obtained resin method, a water content of 10% methanol was heated to dissolve, filtered, cooled to 3 ~ 5 ℃ recrystallized A glucosinolate content can be obtained about 91-93% of the crystalline product of about 2 to 2.5 g, mp 219 ~ 221 ℃.

图3给出A甙的HPLC谱图。 Figure 3 shows the HPLC profile A glycosides.

图1甜菊糖溶液分离前后的HPLC谱图,其中a为原溶液,b为动态分离后的流出液图2母液甜菊糖溶液分离前后的HPLC谱图,其中a为母液糖(4mg/ml),b为树脂分离后的流出液图3重结晶后莱胞迪甙A的HPLC谱图 1 before and after separation HPLC profile of FIG stevioside solution, wherein A is a stock solution, b is the HPLC profile before and after the separation of mother liquor 2 stevioside solution effluent view of the dynamic separation, wherein a is a sugar liquor (4mg / ml), 3 b is a view after recrystallization effluent separation resin Levin extracellular HPLC profile of rebaudioside a

Claims (2)

  1. 1.一种用于分离富集莱鲍迪甙A的大孔吸附树脂,其特征在于它是用下列方法合成:于三口瓶中将工业二乙烯苯50~100克、4-乙烯基吡啶1~15克,过氧化苯甲酰0.5~1.5克,混合均匀,加入混合致孔剂烷基苯与液体石蜡,总重量为50~300克,其中烷基苯与石蜡的重量比为1∶1~10∶1。 1. A method for separation and enrichment rebaudioside A macroporous resin, characterized in that it is synthesized by the following method: To a three-necked flask to 50 to 100 g of commercial divinylbenzene, 4-vinylpyridine 1 to 15 grams of benzoyl peroxide 0.5 to 1.5 grams, mixed, benzene was added and mixed porogen liquid paraffin, a total weight of 50 to 300 grams, wherein the weight ratio of alkyl benzene to paraffin was 1:1 ~ 10:1. 加入NaCl水溶液,使水油相体积比控制在3∶1~5∶1,并加入0.5~1.0%的聚乙烯醇做为表面活性剂。 NaCl solution was added, the aqueous phase volume ratio of the oil control 3:1 ~ 5:1, and 0.5 to 1.0% polyvinyl alcohol as a surfactant. 开动搅拌,控制搅一速度,使小球粒径分布在80~200目之间。 Stirring was stirred at a speed controlled so that the ball size distribution between 80 to 200 mesh. 以5℃/10min的速度匀速缓慢升温至80℃,恒温反应2小时,继续升温至90℃,恒温反应4小时。 At a rate of 5 ℃ / 10min constant speed slowly warmed to 80 ℃, the reaction temperature for 2 hours and continue to heat to 90 ℃, reacting at the temperature for 4 hours. 最后升温至95~100℃煮球2小时,使聚合完全。 Finally, after heating to 95 ~ 100 ℃ cook balls for 2 hours to complete the polymerization. 所得树脂用水充分洗涤,凉干后分别用乙醇、石油醚各抽提8小时,室温下减压干燥备用。 The resulting resin was washed well with water, dry it with ethanol, respectively, each of the petroleum ether extraction for 8 hours and dried under reduced pressure at room temperature for standby. 称取一定量的树脂用适量乙醇溶胀过夜,然后逐渐过渡到水相。 Weighed amount of resin with an appropriate amount of ethanol to swell overnight, then gradually transition to the aqueous phase. 准确量取20ml湿树脂并将其转入内径为0.8~1.0cm树脂柱中,以1BV/h流速使20~30ml稀HCl溶液缓慢流过树脂床,然后水洗至中性。 20ml of wet resin was weighed accurately and transferred into an inner diameter of 0.8 ~ 1.0cm resin column, at 1BV / h flow rate of 20 ~ 30ml so dilute HCl solution was slowly flows through the resin bed, and then washed with water until neutral. 再以1BV/h的流速使20~30ml 1N的NaOH溶液缓慢流过树脂床,水洗至中性备用。 Then at 1BV / h at a flow rate that the solution 20 ~ 30ml 1N NaOH slowly flows through the resin bed, washed with water until neutral standby.
  2. 2.一种以树脂法分离富集莱鲍迪甙A的方法,其特征在于在上述大孔吸附树脂床中,以1BV/h的流速缓慢流过浓度为4mg/ml的甜菊糖水溶液,此时树脂对甜菊糖分子产生选择性吸附作用,检测流出液,380ml开始泄漏,自泄漏点起每20ml为一组分等体积接收流出液,并用HPLC进行监测,直至500ml后流出液中A甙含量小于S甙。 A method for separating resin Enrichment to rebaudioside A, wherein in the above macroporous resin bed to 1BV / h flow rate of slowly flowing at a concentration of 4mg / ml aqueous solution of stevia, this generating selective adsorption of the resin on the molecular stevioside, effluent is monitored, 380 ml of began to leak, the leak from the starting point of each of a volume of 20ml to receive effluent liquid component and the like, and monitored by HPLC, and after 500ml until the effluent a glycoside content S less than glycosides. 合并流出液,浓缩至干燥,得到高A甙含量甜菊糖约200mg。 The combined effluent was concentrated to dryness to obtain a high content of steviol glycosides A to about 200mg. 树脂可用70%乙醇再生,用水洗净后可反复使用。 Resin can be regenerated with 70% ethanol, washed with water can be used repeatedly.
CN 98104776 1998-02-18 1998-02-18 Adsorption resin method for conectrating and separating vegetable baudy glucoside A from stevioside CN1078217C (en)

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